Proteases are enzymes possessing the activity of hydrolyzing peptide bonds in proteins and polypeptides. One subclass of proteases, the metalloproteases are dependent on an integral zinc atom catalysis and often require exogenous calcium for activity. One such enzyme, which has been referenced in the literature as collagenase-like peptidase (EC 3.4.99.31), Pz-peptidase (Barrett (1990) Biol Chem Hoppe-Seyler 371(Supp):311-320) or metalloendopeptidase (EC 3.4.24.15) (Orlowski, et al (1989) Biochem J 261:951-958) cleaves preferentially bonds on the carboxyl side of hydrophobic amino acid residues and is believed to function in the metabolism of bioactive peptides.
Similar enzymatic activity towards collagen sequence-based peptides have been detected in a number of human tissue extracts by various investigators; however, most of the work was confined to the measurement of peptidase activity using collagen sequence-based peptides (Lessley, et al (1985) J Androl 6(6):372-378; Rajabi, et al (1984) Am J Obstet Gynecol 150(7):821-826 and Ito, et al (1977) Clin Chim Acta 78(2):267-270 ). Pierotti, et al (1990) Biochem 29:10323-10329 recently report the molecular cloning and primary structure of rat testes metalloendopeptidase. The enzyme is composed of 645 amino acids with a molecular weight of 72,985 daltons. There does not appear to be any reports that provide identification of human Pz-peptidase, either by partial purification and characterization, or by using a battery of substrates or inhibitor profiles.
Recently, investigators have preliminarily identified a proteolytic activity from human brain of about 68,000 daltons that is capable of cleaving between the Met and Asp residues of a small synthetic peptide HSEVKMDAEF, which corresponds to amino acids 592 through 600 in the .beta.-amyloid precursor protein ("APP") (Abraham, et al (1990) Neurobiol Aging 11A:303) with an N-terminal His. When this peptide, having an .sup.125 I radioiodinated His residue, is incubated with "brain protease" fractions, fragments are generated and separated by thin layer chromatography (TLC). The N-terminal fragments were detected by exposure of the TLC plate to film (Abraham, et al (1991) Biochem Biophys Res Comm. 174: 790-796). The cleavage pattern obtained with the brain protease preparation was primarily at three sites, between the Lys-Met, Met-Asp, and Asp-Ala, with some cleavage obtained at His-Ser. The peptide cleavage was inhibited by diisopropylfluorophosphate (DFP), .alpha..sub.1 -antichymotrypsin, and protease nexin II, all of which only inhibit serine proteases. Based on chemical crosslinking studies with the iodinated peptide substrate, two bands, one at approximately 68,000 daltons and another at approximately 30,000 daltons, are suggested to be candidates for the protease(s) in the preparation.
In more recent presentations (Abraham, et al (1991) J Cell Biochem Suppl. 15G:115; Abraham, et al (1991) J Neurochem 57 (Suppl.):S109), these investigators claim at least two different proteases in the preparation, one being the previously described calcium-dependent serine protease, and the other a cysteine metalloprotease. To date, no structure or characterization or any of these proteases has been presented.
APP is a membrane-spanning glycoprotein that is expressed in many mammalian tissues and cell lines and is encoded by a gene that, in humans, is found on chromosome 21. The .beta.-amyloid core protein, referred to as the .beta.- or A4 peptide, is an approximately 39-42 amino acid long peptide fragment of APP, and is the major component of the myriad amyloid deposits that accumulate extracellularly in the brains of patients with Alzheimer's disease (AD) or form the cerebrovascular amyloid in associated blood vessels.
There are at least three forms of the precursor protein: APP695 (Kang, et al (1987) Nature 325:733); APP751 (Ponte, et al (1988) Nature 331:525); and APP770 (Kitaguchi, et al (1988) Nature 331:530) which refer to the number of amino acids in the primary protein transcript. All of these forms contain the .beta.-peptide sequence, which starts 28 amino acids N-terminal to the beginning of the putative transmembrane region, and ends approximately 14 amino acids in the transmembrane region. The numbering of amino acids as used herein corresponds to that used for APP695.
Recent work on the metabolism of the APP in cell culture has clearly shown that after intracellular maturation of the full transmembrane form of the protein, there is a specific proteolytic processing event which leads to extracellular secretion of a large N-terminal region, and leaves behind in cell membranes a small, C-terminal fragment reactive with antisera to the carboxyl end of the APP (Oltersdorf, et al (1990) J Biol Chem 265:4492). The size of this C-terminally reactive fragment made it likely that it contains the entire .beta.-peptide. However, characterization by direct protein sequencing of the N-terminal of this fragment showed that it starts at Leu17 of the .beta.-amyloid core peptide where Asp597 of APP695 is counted as Aspl of the .beta.-peptide (Esch, et al (1990) Science 248:1122). Characterization of the soluble secreted form by isolation of the peptide containing its C-terminal region also clearly showed that it ends at Gln15. Thus, in this normal processing pathway, the transmembrane form of APP is cleaved inside the .beta.-peptide (either before or after Lys16, which is missing; presumably, it is taken off either by a carboxypeptidase or an aminopeptidase activity post-cleavage), and thus this pathway precludes the formation or deposition of the .beta.-peptide. It also follows then that an alternative proteolytic pathway must exist for generation of the .beta.-peptide.
The most likely characteristic of such a pathway would be a proteolytic cleavage between Met596 and Asp597, since protein sequencing of either senile (core) or vascular amyloid always starts at this aspartic acid residue, although there has been reported to be a ragged N-terminus for core amyloid (Masters, et al (1985) Proc Natl Acad Sci U.S.A. 82:4245). The preliminary work reported by Abraham et al (1990) supra, provides some insight as to how APP might be proteolytically processed to release the .beta.-amyloid core protein.
Identification of mammalian proteases that are capable of cleavage at this site is essential in order to screen for inhibitors of such cleavage. Such inhibitors would be useful for therapeutic intervention in AD.
Cell culture models of the blood brain barrier may be used for the design of drug delivery systems for the inhibitors of the present invention. Such cell culture models are disclosed in U.S. Pat. No. 90/05106, filed 13 Sep. 1990 and U.S. Pat. No. 90/05105, filed 13 Sep. 1990.